HEMA-Lysine-Based Cryogels for Highly Selective Heparin Neutralization.

Unfractionated heparin (UFH) and its low-molecular-weight fragments (LMWH) are widely used as anticoagulants for surgical procedures and extracorporeal blood purification therapies such as cardiovascular surgery and dialysis. The anticoagulant effect of heparin is essential for the optimal execution of extracorporeal blood circulation. However, at the end of these procedures, to avoid the risk of bleeding, it is necessary to neutralize it. Currently, the only antidote for heparin neutralization is protamine sulphate, a highly basic protein which constitutes a further source of serious side events and is ineffective in neutralizing LMWH. Furthermore, dialysis patients, due to the routine administration of heparin, often experience serious adverse effects, among which HIT (heparin-induced thrombocytopenia) is one of the most severe. For this reason, the finding of new heparin antagonists or alternative methods for heparin removal from blood is of great interest. Here, we describe the synthesis and characterization of a set of biocompatible macroporous cryogels based on poly(2-hydroxyethyl methacrylate) (pHEMA) and L-lysine with strong filtering capability and remarkable neutralization performance with regard to UFH and LMWH. These properties could enable the design and creation of a filtering device to rapidly reverse heparin, protecting patients from the harmful consequences of the anticoagulant.

Towards a reference material for microplastics' number concentration-case study of PET in water using Raman microspectroscopy.

Increasing demand for size-resolved identification and quantification of microplastic particles in drinking water and environmental samples requires the adequate validation of methods and techniques that can be used for this purpose. In turn, the feasibility of such validation depends on the existence of suitable certified reference materials (CRM). A new candidate reference material (RM), consisting of polyethylene terephthalate (PET) particles and a water matrix, has been developed. Here, we examine its suitability with respect to a homogeneous and stable microplastic particle number concentration across its individual units. A measurement series employing tailor-made software for automated counting and analysis of particles (TUM-ParticleTyper 2) coupled with Raman microspectroscopy showed evidence of the candidate RM homogeneity with a relative standard deviation of 12% of PET particle counts involving particle sizes >30 µm. Both the total particle count and the respective sums within distinct size classes were comparable in all selected candidate RM units. We demonstrate the feasibility of production of a reference material that is sufficiently homogeneous and stable with respect to the particle number concentration.

Conjugation to gold nanoparticles of methionine gamma-lyase, a cancer-starving enzyme. Physicochemical characterization of the nanocomplex for prospective nanomedicine applications.

The pyridoxal 5'-dependent enzyme methionine γ-lyase (MGL) catalyzes the degradation of methionine. This activity has been profitable to develop an antitumor agent exploiting the strict dependence of most malignant cells on the availability of methionine. Indeed, methionine depletion blocks tumor proliferation and leads to an increased susceptibility to anticancer drugs. Here, we explore the conjugation of MGL to gold nanoparticles capped with citrate (AuNPs) as a novel strategy to deliver MGL to cancer cells. Measurements of Transmission Electron Microscopy, Dynamic Light Scattering, Asymmetrical Flow Field-Flow Fractionation, X-ray Photoelectron Spectroscopy, and Circular Dichroism allowed to achieve an extensive biophysical and biochemical characterization of the MGL-AuNP complex including particle size, size distribution, MGL loading yield, enzymatic activity, and impact of gold surface on protein structure. Noticeably, we found that activity retention was improved over time for the enzyme adsorbed to AuNPs with respect to the enzyme free in solution. The acquired body of knowledge on the nanocomplex properties and this encouraging stabilizing effect upon conjugation are the necessary basis for further studies aimed at the evaluation of the therapeutic potential of MGL-AuNP complex in a biological milieu.

Differences in Physico-Chemical Properties and Immunological Response in Nanosimilar Complex Drugs: The Case of Liposomal Doxorubicin.

This study aims to highlight the impact of physicochemical properties on the behaviour of nanopharmaceuticals and how much carrier structure and physiochemical characteristics weigh on the effects of a formulation. For this purpose, two commercially available nanosimilar formulations of Doxil and their respective carriers were compared as a case study. Although the two formulations were "similar", we detected different toxicological effects (profiles) in terms of in vitro toxicity and immunological responses at the level of cytokines release and complement activation (iC3b fragment), that could be correlated with the differences in the physicochemical properties of the formulations. Shedding light on nanosimilar key quality attributes of liposome-based materials and the need for an accurate characterization, including investigation of the immunological effects, is of fundamental importance considering their great potential as delivery system for drugs, genes, or vaccines and the growing market demand.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 µg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 µg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Investigating the Cellular Uptake of Model Nanoplastics by Single-Cell ICP-MS.

A synthetic route to producing gold-doped environmentally relevant nanoplastics and a method for the rapid and high-throughput qualitative investigation of their cellular interactions have been developed. Polyethylene (PE) and polyvinyl chloride (PVC) nanoparticles, doped with ultrasmall gold nanoparticles, were synthesized via an oil-in-water emulsion technique as models for floating and sedimenting nanoplastics, respectively. Gold nanoparticles were chosen as a dopant as they are considered to be chemically stable, relatively easy to obtain, interference-free for elemental analysis, and suitable for bio-applications. The suitability of the doped particles for quick detection via inductively coupled plasma mass spectrometry (ICP-MS), operating in single-cell mode (scICP-MS), was demonstrated. Specifically, the method was applied to the analysis of nanoplastics in sizes ranging from 50 to 350 nm, taking advantage of the low limit of detection of single-cell ICP-MS for gold nanoparticles. As an initial proof of concept, gold-doped PVC and PE nanoplastics were employed to quantify the interaction and uptake of nanoplastics by the RAW 264.7 mouse macrophage cell line, using scICP-MS and electron microscopy. Macrophages were chosen because their natural biological functions would make them likely to internalize nanoplastics and, thus, would produce samples to verify the test methodology. Finally, the method was applied to assess the uptake by CaCo-2 human intestinal cells, this being a more relevant model for humanexposure to those nanoplastics that are potentially available in the food chain. For both case studies, two concentrations of nanoplastics were employed to simulate both standard environmental conditions and exceptional circumstances, such as pollution hotspot areas.

Characterization of microparticles derived from waste plastics and their bio-interaction with human lung A549 cells.

Microplastics (MPs) represent a worldwide emerging relevant concern toward human and environmental health due to their intentional or unintentional release. Human exposure to MPs by inhalation is predicted to be among the most hazardous. MPs include both engineered, or primary MPs, and secondary MPs, materials obtained by fragmentation from any plastic good. The major part of the environmental MPs is constituted by the second ones that are irregular in size, shape and composition. These features make the study of the biological impact of heterogenous MPs of extremely high relevance to better estimate the real toxicological hazards of these materials on human and environmental organisms. The smallest fractions of plastic granules, relying on the micron-sized scale, can be considered as the most abundant component of the environmental MPs, and for this reason, they are typically used to perform toxicity tests using in vitro systems representative of an inhalation exposure scenario. In the present work, MPs obtained from industrial treatment of waste plastics (wMPs < 50 µm) were investigated, and after the physico-chemical characterization, the cytotoxic, inflammatory and genotoxic responses, as well as the modality of wMPs interactions with alveolar lung cells, were determined. Obtained results indicated that, at high concentrations (100 µg/ml) and prolonged exposure time (48 h), wMPs affect biological responses by inducing inflammation and genotoxicity, as a result of the cell-wMP interactions, also including the uptake of the smaller particles.

Detecting Micro- and Nanoplastics Released from Food Packaging: Challenges and Analytical Strategies.

Micro- and nanoplastic (pMP and pNP, respectively) release is an emerging issue since these particles constitute a ubiquitous and growing pollutant, which not only threatens the environment but may have potential consequences for human health. In particular, there is concern about the release of secondary pMP and pNP from the degradation of plastic consumer products. The phenomenon is well-documented in relation to plastic waste in the environment but, more recently, reports of pMP generated even during the normal use of plastic food contact materials, such as water bottles, tea bags, and containers, have been published. So far, a validated and harmonized strategy to tackle the issue is not available. In this study, we demonstrate that plastic breakdown to pMP and pNP can occur during the normal use of polyethylene (PE) rice cooking bags and ice-cube bags as well as of nylon teabags. A multi-instrumental approach based on Raman microscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and particular attention on the importance of sample preparation were applied to evaluate the chemical nature of the released material and their morphology. In addition, a simple method based on Fourier transform infrared (FT-IR) spectroscopy is proposed for pNP mass quantification, resulting in the release of 1.13 ± 0.07 mg of nylon 6 from each teabag. However, temperature was shown to have a strong impact on the morphology and aggregation status of the released materials, posing to scientists and legislators a challenging question: are they micro- or nanoplastics or something else altogether?

Preparation of a reference material for microplastics in water-evaluation of homogeneity.

Validation of analytical methods for measurements of microplastics (MP) is severely hampered because of a general lack of reference materials, RM. There is a great need to develop such reference materials. This study presents a concept of three-component kit with immobilised MP in solid NaCl, a surfactant and clean water that can be applied for the production of many types of MP RMs. As proof of concept, an RM for polyethylene terephthalate (PET) particles in water was prepared and evaluated for its homogeneity. The particles ranged from 30 µm (Feretmin) to about 200 µm adapted by wet sieving. A specific number of PET particles were immobilized in about 0.29 g of solid NaCl by freeze-drying 1 mL of a NaCl suspension. By using manual and automated counting, twenty reconstituted 1-L water samples were evaluated for homogeneity with respect to number of PET particles from 30 µm to > 200 µm/L of water. The number of particles was 730 ± 120 (mean ± one standard deviation (SD); n = 10) and 865 ± 155 particles (n = 10) obtained by optical microscopy in two independent laboratories. This corresponded to relative SDs of 16.4 and 17.9% and a mean of 797 ± 151 particles (18.9% RSD, for n = 20). Homogeneity studies of the NaCl carrier without reconstitution resulted in 794 ± 60 particles (7.5% RSD). The homogeneity of PET in the salt carrier was also evaluated directly with respect to mass of PET per vial using an ultra-micro balance. An average mass of 293 ± 41 µg of PET was obtained (14, % RSD for n = 14). Micrographs were recorded to demonstrate the absence of major sources of contamination of the RM components. Information about the particle size distribution and particle shapes was obtained by laser diffraction (LD) and dynamic image analysis (DIA). In addition, the identity of the PET polymer was confirmed by Raman and FT-IR spectroscopy. The RM was developed for a large-scale inter-laboratory comparison of PET particles in water (ILC). Based on the homogeneity results, the material was found to be sufficiently homogeneous to be of meaningful use in the ILC. In a 3-day process, more than 500 samples of PET particles in the NaCl carrier were prepared with good potential for further upscaling with respect to the number of vials or with other kinds of polymers. The stability of PET was not evaluated but it was deemed to be stable for the duration of the ILC.

Microplastics from miscellaneous plastic wastes: Physico-chemical characterization and impact on fish and amphibian development.

Microplastic pollution represents a global problem with negative impacts on aquatic environment and organisms' health. To date, most of the laboratory toxicological studies on microplastics (MPs) have made use of single commercial micro and nano-polymers, which do not reflect the heterogeneity of environmental MPs. To improve the relevance of the hazard assessment, micrometer-sized plastic particles of miscellaneous non-reusable waste plastics, with size <100 µm and <50 µm (waste microplastics, wMPs), were characterized by microscopic and spectroscopic techniques and tested on developing zebrafish and Xenopus laevis by FET and FETAX assays respectively. Moreover, the modalities of wMP interaction with the embryonic structures, as well as the histological lesions, were explored by light and electron microscopy. We have shown that wMPs had very heterogeneous shapes and sizes, were mainly composed of polyethylene and polypropylene and contained metal and organic impurities, as well as submicrometric particle fractions, features that resemble those of environmental occurring MPs. wMPs (0.1-100 mg/L) caused low rate of mortality and altered phenotypes in embryos, but established species-specific biointeractions. In zebrafish, wMPs by adhering to chorion were able to delay hatching in a size and concentration dependent manner. In Xenopus embryos, which open stomodeum earlier than zebrafish, wMPs were accumulated in intestinal tract, where produced mechanical stress and stimulated mucus overproduction, attesting an irritation response. Although wMP biointeractions did not interfere with morphogenesis processes, further studies are needed to understand the underlying mechanisms and long-term impact of these, or even smaller, wMPs.

Personalised Profiling of Innate Immune Memory Induced by Nano-Imaging Particles in Human Monocytes.

Engineered nanoparticles used for medical purposes must meet stringent safety criteria, which include immunosafety, i.e., the inability to activate possibly detrimental immune/inflammatory effects. Even medical nanomaterials devoid of direct immunotoxic or inflammatory effects may have an impact on human health if able to modify innate memory, which is the ability to "prime" future immune responses towards a different, possibly more detrimental reactivity. Although innate memory is usually protective, anomalous innate memory responses may be at the basis of immune pathologies. In this study, we have examined the ability of two nanomaterials commonly used for diagnostic imaging purposes, gold and iron oxide nanoparticles, to induce or modulate innate memory, using an in vitro model based on human primary monocytes. Monocytes were exposed in culture to nanoparticles alone or together with the bacterial agent LPS (priming phase/primary response), then rested for six days (extinction phase), and eventually challenged with LPS (memory/secondary response). The memory response to the LPS challenge was measured as changes in the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra), as compared to unprimed monocytes. The results show that both types of nanoparticles can have an effect in the induction of memory, with changes observed in the cytokine production. By comparing nanomaterials of different shapes (spherical vs. rod-shaped gold particles) and different size (17 vs. 22 nm diameter spherical iron oxide particles), it was evident that innate memory could be differentially induced and modulated depending on size, shape and chemical composition. However, the main finding was that the innate memory effect of the particles was strongly donor-dependent, with monocytes from each donor showing a distinct memory profile upon priming with the same particles, thereby making impossible to draw general conclusions on the particle effects. Thus, in order to predict the effect of imaging nanoparticles on the innate memory of patients, a personalised profiling would be required, able to take in consideration the peculiarities of the individual innate immune reactivity.

New Detection Platform for Screening Bacteria in Liquid Samples.

The development of sensitive methods for the determination of potential bacterial contamination is of upmost importance for environmental monitoring and food safety. In this study, we present a new method combining a fast pre-enrichment step using a microporous cryogel and a detection and identification step using antimicrobial peptides (AMPs) and labelled antibodies, respectively. The experimental method consists of: (i) the capture of large amounts of bacteria from liquid samples by using a highly porous and functionalized cryogel; (ii) the detection and categorisation of Gram-positive and Gram-negative bacteria by determining their affinities toward a small set of AMPs; and (iii) the identification of the bacterial strain by using labelled detection antibodies. As proof of concept, the assessment of the three steps of the analysis was performed by using Escherichia coli and Bacillus sp. as models for Gram-negative and Gram-positive bacteria, respectively. The use of AMPs with broad specificity combined with labelled antibodies enabled the detection and potential categorization of a large spectrum of unknown or unexpected bacteria.

Detection of Metal-Doped Fluorescent PVC Microplastics in Freshwater Mussels.

The large-scale production of plastic and the resulting release of waste is leading to a huge accumulation of micro-sized particles in the environment that could have an impact on not only aquatic organisms but also on humans. Despite the extensive literature on the subject, there is still an insufficient harmonization of methodologies for the collection and analysis of microplastics (MPs) in complex matrices; especially for high density polymers; such as polyvinyl chloride (PVC), which tend to sink and accumulate in sediments, becoming available to benthonic organisms. In this article, mussels have been chosen as model for microplastic accumulation due to their extensive filtering activity and their wide distribution in both fresh and salt water basins. To facilitate the identification and quantification of microplastics taken up by mussels, novel fluorescent and metal-doped PVC microplastics (PVC-Platinum octaethylporphyrin (PtOEP) MPs in the size range of 100 µm) have been synthesized and characterized. For the analysis of the mussels following exposure, an enzymatic protocol using amylase, lipase, papain, and SDS for organic material digestion and a sucrose-ZnCl2 density gradient for the selective separation of ingested microplastics has been developed. The final identification of MPs was performed by fluorescence microscopy. This work can greatly benefit the scientific community by providing a means to study the behavior of PVC MPs, which represent an example of a very relevant yet poorly studied high density polymeric contaminant commonly found in complex environmental matrices.

Synthesis of Citrate-Stabilized Silver Nanoparticles Modified by Thermal and pH Preconditioned Tannic Acid.

Silver nanoparticles (AgNPs) may be synthesized by many different methods, with those based on the thermal reduction of silver salts by citric acid or citric acid/tannic acid being amongst the most commonly used. These methods, although widely used and technically simple, can produce particles in which the size, polydispersivity and morphology can vary greatly. In this work nearly mono-dispersed spherical AgNPs have been synthesized via a one-step reduction method by using sodium citrate and varying quantities of Tannic Acid (TA), which was thermally conditioned prior to use in the growth process. It was found that the final size can be further tailored by controlling the amount of TA and the thermal conditioning of the TA at 60 °C at different time points, which changes the size and polydispersivity of AgNPs. To better understand the origin of this effect, optical spectroscopic analysis and 1H NMR of the TA following mild thermal conditioning of the solution have been done. Comparison of thermally conditioned TA and TA exposed to basic pH shows that similar chemical modifications occur and consequently produce similar effects on growth when used in the synthesis of AgNPs. It is proposed that thermal preconditioning of the TA introduces either chemical or structural changes, which decrease the final particle size under a given total silver content.

Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine.

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials.

Dark Field Microscopy-Based Biosensors for the Detection of E. coli in Environmental Water Samples.

Development of sensitive methods for the determination of E. coli bacteria contamination in water distribution systems is of paramount importance to ensure the microbial safety of drinking water. This work presents a new sensing platform enabling the fast detection of bacteria in field samples by using specific antibodies as the biorecognition element and dark field microscopy as the detection technique. The development of the sensing platform was performed using non-pathogenic bacteria, with the E. coli DH5α strain as the target, and Bacillus sp. 9727 as the negative control. The identification of the captured bacteria was made by analyzing the dark field microscopy images and screening the detected objects by using object circularity and size parameters. Specificity tests revealed the low unspecific attachment of either E. coli over human serum albumin antibodies (negative control for antibody specificity) and of Bacillus sp. over E. coli antibodies. The system performance was tested using field samples, collected from a wastewater treatment plant, and compared with two quantification techniques (i.e., Colilert-18 test and quantitative polymerase chain reaction (qPCR)). The results showed comparable quantification capability. Nevertheless, the present method has the advantage of being faster, is easily adaptable to in-field analysis, and can potentially be extended to the detection of other bacterial strains.

SUNSPACE, A Porous Material to Reduce Air Particulate Matter (PM).

The World Health Organization reports that every year several million people die prematurely due to air pollution. Poor air quality is a by-product of unsustainable policies in transportation, energy, industry, and waste management in the world's most crowded cities. Particulate matter (PM) is one of the major element of polluted air. PM can be composed by organic and inorganic species. In particular, heavy metals present in PM include, lead (Pb), mercury (Hg), cadmium, (Cd), zinc (Zn), nickel (Ni), arsenic (As), and molybdenum (Mo). Currently, vegetation is the only existing sustainable method to reduce anthropogenic PM concentrations in urban environments. In particular, the PM-retention ability of vegetation depends on the surface properties, related to the plant species, leaf and branch density, and leaf micromorphology. In this work, a new hybrid material called SUNSPACE (SUstaiNable materials Synthesized from by-Products and Alginates for Clean air and better Environment) is proposed for air PM entrapment. Candle burning tests are performed to compare SUNSPACE with Hedera Helix L. leafs with respect to their efficacy of reducing coarse and fine PM. The temporal variation of PM10 and PM2.5 in presence of the trapping materials, shows that Hedera Helix L. surface saturates more rapidly. In addition, the capability of SUNSPACE in ultrafine PM trapping is also demonstrated by using titanium dioxide nanoparticles with 25 nm diameter. Scanning electron microscope (SEM) and Transmission electron microscope (TEM) images of SUNSPACE after entrapment tests highlight the presence of collected nanoparticles until to about 0.04 mm in depth from the sample surface. N2 physisorption measurements allow to demonstrate the possibility to SUNSPACE regeneration by washing.

Proteomics study of silver nanoparticles on Caco-2 cells.

Silver nanoparticles (AgNPs) have been incorporated into several consumer products. While these advances in technology are promising and exciting, the effects of these nanoparticles have not equally been studied. Due to the size, AgNPs can penetrate the body through oral exposure and reach the gastrointestinal tract. The present study was designed as a comparative proteomic analysis of Caco-2 cells, used as an in vitro model of the small intestine, exposed to 30 nm citrate stabilized-silver nanoparticles (AgNPs) for 24 or 72 h. Using two complementary proteomic approaches, 2D gel-based and label-free mass spectrometry, we present insight into the effects of AgNPs at proteins level. Exposure of 1 or 10 µg/mL AgNPs to Caco-2 cells resulted in 56 and 88 altered proteins at 24 h and 72 h respectively, by 2D gel-based technique. Ten of these proteins were found to be common between the two time-points. Using label-free mass spectrometry technique, 291 and 179 altered proteins were found at 24 h and 72 h, of which 24 were in common. Analysis of the proteomes showed several major biological processes altered, from which, cell cycle, cell morphology, cellular function and maintenance were the most affected.

Label-Free Biosensor Detection of Endocrine Disrupting Compounds Using Engineered Estrogen Receptors.

Endocrine Disrupting Compounds (EDCs) are chemical substances shown to interfere with endogenous hormones affecting the endocrine, immune and nervous systems of mammals. EDCs are the causative agents of diseases including reproductive disorders and cancers. This highlights the urgency to develop fast and sensitive methods to detect EDCs, which are detrimental even at very low concentrations. In this work, we propose a label-free surface plasmon resonance (SPR) biosensor method to detect specific EDCs (17 ß-estradiol (E2), ethinyl-estradiol, 4-nonylphenol, tamoxifen) through their binding to estrogen receptor alpha (ERα). We show that the use of rationally designed ERα (as bio-recognition element) in combination with conformation-sensitive peptides (as amplification agent, resulting in increased responses) enables the detection of low parts per billion (ppb) levels of E2. As a proof of concept, this bioassay was used to detect E2 in (spiked) real water samples from fish farms, rivers and the sea at low ppb levels after concentration by solid phase extraction. In addition, the present SPR assay that combines a conformation-sensitive peptide with an array of ERα mutants is very promising for the assessment of the risk of potential estrogenic activity for chemical substances.

Influence of different cleaning processes on the surface chemistry of gold nanoparticles.

In this paper, the authors have investigated the effects of different cleaning methods (centrifugation and dialysis) on the surface chemistry and composition of 15 nm sodium citrate stabilized gold nanoparticles. The nuclear magnetic resonance (NMR) results indicate that three centrifugation cycles are sufficient to remove most of the citrate molecules, while centrifuged liquid sedimentation and dynamic light scattering data reveal some degree of nanoparticle aggregation when three centrifugation cycles are exceeded. Regarding the dialysis procedure, NMR analysis demonstrated that after nine cleaning cycles, the citrate concentration is comparable to that measured after the first centrifugation (about 6 × 10-4 M) but with an increase in the dispersion polydispersivity index as determined by dynamic light scattering. X-ray photoelectron spectroscopy results support the NMR findings and revealed a major hydrocarbon contamination after the nanoparticles cleaning process. The impact of cleaning on surface functionalization was tested using 1H,1H,2H,2H-perfluorodecanethiol hydrophobic thiols (PFT) to test thiol-citrate substitution. After 24 h exposure, the PFT coverage was less than 0.6 monolayer (ML) for both pristine nanoparticles and particles after three dialysis cycles, but about 0.8 ML after two centrifugation washes.